Project 1 is Investigating the mechanism of electrical rhythmicity in Gl muscles. Phasic contractions in the Gl tract are timed and graded in amplitude by electrical slow waves generated by interstitial cells of Cajal (ICC). A variety of human motility disorders have been associated with defects in ICC function, dysrhythmias or loss of slow waves. Knowledge of the pacemaker mechanism may allow development of novel therapies to enhance or restore slow wave activity. We have isolated ICC from mouse colon and small intestine, purified these cells by fluorescence activated cell sorting and performed next generation (deep) sequencing to characterize the transcriptomes of these cells. Using this extensive molecular library of genes expressed in ICC, we have conceived a model of the 'pacemakerzome' in ICC that is based on functional needs for pacemaking and predominent genes expressed. We have determined a collection of essential proteins that are capable of maintaining the ionic gradients and requirements of the pacemaker units of ICC. Experiments proposed will test relative contributions of identified ion channels and transporters that facilitate and sustain pacemaker activity in ICC. We will study the primary pacemaker current responsible for spontaneou transient inward currents (STICs) that are the basic events responsible for pacemkaer activity, and the slow wave currents (summed STICs) that are responsible for slow waves in intact muscles. The splice variants of Ca2+-activated Cl- channels (CaCC) responsible for STICs and slow wave currents will be studied to understand differences in slow wave characteristics and pharmacology in different regions of the Gl tract, Ca2+ entry mechanisms that support rhythmic openings of CaCC during pacemaker currents will be determined, and transport proteins responsible for restoration of critical ionic gradients will be characterized. Deep sequencing provided important clues about the molecular identity of gene products responsible for these functions, and this information will be exploited for studies of transgenic mice to investigate many of the questions to be addressed during the next funding period. RELEVANCE (See instructions): Interstitial cells of Cajal generate pacemaker activity in the Gl tract that is responsible for peristaltic and segmental contractions in motility. Defects in pacemaker activity lead to disordered motility and dysregulated transit of food and nutrients in human patients. Understanding how pacemaker cells work will provide new ideas about how to control Gl motility therapeutically.